Copolymers of acrylamide gas

ABSTRACT

Water insoluble biologically active proteins are bound on novel copolymers consisting of (a) acrylamide (b) ethylene-maleic acid or its anhydride and/or (c) maleic acid and/or its anhydride and (d) N,N&#39;&#39;-methylene-bis-acrylamide or ethylene diacrylate in a weight ratio a:b:c:d of 3 : 0.5 - 1.5 : 0.05 - 4 : 0.075 - 0.9, the weight ratio a : (b + c) being not more than 4, to provide compositions which are outstanding in their ability to yield up the bound protein, and with which precise substrate reactions can be carried out.

United States Patent [191 Jaworek'et al.

[ 1 Jan. 16,1973

1 1 COPOLYMERS OF ACRYLAMIDE GAS [75] Inventors: Dieter Jaworek, Tutzing/Obb.; Wolfgang Gruber, Garatshausen; Hans Ulrich Bergmeyer, Tutzing/Obb., all of Germany [73] Assignee: Boehringer Mannheim GrnbH,

Mannheim, Germany [22] Filed: Feb. 6, 1970 [21] App]. No.: 9,442

[30'] Foreign Application Priority Data Feb. 19, 1969 Germany ...P 19 08 290.7

[52] U.S. CL. .,.260/878 R, 195/63, 210/24,

260/8, 260/78.5 BB [51] lnt. Cl. ..C08f 15/40, C08f 29/34 [58] Field of Search ..260/78.5 R. 78.5 T, 878 R.

[56] References Cited UNITED STATES PATENTS 3,398,092 Fields et al. ..2l0/24 3,488,215 l/1970 Shepherd et al ..1 17/124 1 3,450,680 6/1969 Jursich et a1. ..260/78.5 2,977,334 3/1961 Zopf et al. ..260/27 Primary Examiner-James A. Seidleck Assistant Examiner lohn Kight, lll Attorney-Burgess, Dinklage & Sprung 57 ABSTRACT 8 Claims, No Drawings COPOLYMERS OF ACRYLAMIDE GAS The present invention is concerned with water insoluble proteins bound to new polymeric carrier materials and is also concerned with the preparation of such bound protein materials and with the polymeric carrier materials and the production of the polymeric carrier materials.

In principle, a differentiation should be made between proteins which are inherently insoluble and those which are insoluble in bound form.

It is known that insoluble proteins can'be formed by condensation, coupling and polymerization reactions with the protein itself. The proteins obtained in this manner are products of uncontrollable reactions; in other words, products with a definite constitution cannot be obtained. A large part of the protein becomes denatured by the severe reaction conditions of the polymerization and of the coupling with, for example, bis-diazo compounds and by the action of the components used for the condensation, such as formaldehyde, ethyl chloroformate and the like, and thus is biologically inactivated. For most fields of use of insoluble proteins, such denatured material is useless.

As insoluble, biologically-active proteins, only those proteins which are fixed on carrier materials are of importance since they are materials of definite constitution which can generally be used universally as enzymes, inhibitors, antigens or anti-bodies or as proteins.

The binding of the proteins to the carrier materials can take place in a heteropolar or homopolar manner. The heteropolar bound proteins are of lesser interest since the proteins can be lost by elution from the carrier materials, in amounts depending upon the ion concentration and pH value of solutions with which they are contacted.

The following methods have been used for fixing proteins to insoluble carriers by means of reactive groups:

1. Reaction of cellulose with p-nitrobenzyl chloride to give the corresponding cellulose derivative, which is reduced to the corresponding amino derivative and diazotized and then coupled with proteins;

2. The reactive groups described in (l) are replaced by m-aminobenzyloxymethyl radicals;

3. Polyamino-polystyrene, which is a completely synthetic carrier, is reacted with proteins;

4. d,1-p-aminophenyl-alanine, d,l-leucine and similar amino acids are polycondensed to give a synthetic carrier, the diazotized amino groups of which are coupled with proteins;

5. By means of methods known from peptide chemistry, carboxymethyl-cellulose is reacted with proteins using dicyclohexyl carbodiimide as condensation agent;

6. From carboxymethyl-cellulose there is produced, via the hydrazide, the corresponding azide which is reacted with proteins;

7. Proteins have been bound with bromoacetyl-cellulose;

8. From isothiocyanate derivatives of the dextran gel known under the Registered Trade Mark Sephadex and cellulose, there have been produced water-insoluble enzyme compounds with proteins;

9. Under the conditions of acrylarnide polymerization, proteins can be fixed in readily swellable material. The enzyme is thereby bound radically on the chains;

10. Furthermore, proteins have also been bound to ethylene-maleic acid anhydride (EMA);

l 1. Proteins have also been bound to styrene maleic anhydride copolymers. I if The previously known methods are not satisfactory and are not of general applicability for proteins. Thus, less than 50 percent of protein is bound by methods 1, 2 and 4, and a greater part of the protein loses its biological activity. Method 3 has proven to be even less suitable.

Method 5 can only be carried out in a non-aqueous medium and, therefore, can only be used for some antibody and low molecular weight inhibitors.

According to method 6, only about 10 percent of the protein used is bound, only 10 43 percent of which retains its enzymatic activity.

Method 7 gives a high yield of bound protein percent) but for many proteins, the reaction conditions used result in a loss of biological activity.

In the case of method 8, only a very small percentage of the protein used is actively bound to the gel.

According to method 9, only 18 20 percent of the protein used is bound, only a few per cent of which can evolve an activity against substrates. High molecular weight substrates, such as hemoglobin, only reacted to an extent of l 2%.

At the moment, method l0 gives the most satisfactory results. It is used for antigens, inhibitors and enzymes. The binding reaction takes place under mild conditions in an aqueous medium; about 50 60 percent of the protein can be fixed under optimum conditions.

However, the starting material used is very nonuniform: it contains lower molecular weight fractions, some of which are soluble, and higher molecular weight fractions, which are insoluble, which cannot be separated by sieving or by other means. Therefore, only up to 60 percent of the protein is bound to the insoluble carrier, the remainder being lost as soluble material. However, the bound protein is still not satisfactory.

In the binding of the protein to the carrier (EMA), the protein fulfills the function of a comonomer; it can be bound not only at one position but also at several positions, i.e., it may be cross-linked. Depending upon the protein concentration, more or less readily swellable protein-carrier materials are obtained. The protein is thereby present in a network in covalent bound form so that diffusion plays an important part in the case of the use of this carrier and it is only partially accessible to a substrate. This disadvantage is not even overcome by the addition of cross-linking agents, such as hexamethylene-diamine or the like. Therefore, only about 50 percent of the bound protein can be reached by low molecular weight substrates, so that the total activity thereof does not amount to more than 30 percent. In the case of high molecular weight substrates, an activity of only 10 percent or less has been found.

An additional disadvantage of this material is that it is of varyingly difficult filterability, i.e., it can, for example, be flocculent, which differs from one protein to another, and is also dependent upon the protein concentration. Thus, there is no assurance that the bound protein produced therewith can be packed into columns.

Styrene-maleic anhydride polymers used according to method 1 1 can be used for the removal of proteins, for example, in the purification of drinking water and the like. These carriers have, similarly to the polyamino-styrenes, a decidedly lyophilic character. Proteins cannot be bound to their structure without loss of activity and, when such polymers are bound with proteins, they cannot be lyophilized.

The functional groups of the known carriers which are not bound to protein carry, in the appropriate pl-l ranges, positively charged Nl-l groups in the case of methods 1, 2, 3 and 4 and negatively charged COO-- groups in the case of methods 6, 8 and 10. In the case of method 10, COO- groups are additionally formed by the bonding of protein to the cyclic anhydride groups of the polymer. This polyvalent character of the protein carriers causes adsorption of the matrix, in a manner similar to ion exchangers, increased inactivation or denaturing of the protein structure and displacement of the pH, optima in the case of bound enzymes.

There is, therefore, a great interest in the provision In the production of the copolymer, the ratio of acrylamide to maleic acidor ethylene maleic acid or to the corresponding anhydrides can be varied within the given ranges without substantially affecting the necessary amount of cross-linking agent (d). Thus, acrylamide can be polymerized with maleic acid or its anhydride in a ratio by weight of 3 0.1 to 4 without the weight ratio of acrylamide to cross-linking agent hereby being substantially influenced. However, as indicated above, in the case of the use of ethylene-maleic acid or of its anhydride as comonomer, the limits are narrower. In this case, the content of cross-linking agent governs the pore size and strength, as well as the of a suitable carrier for water-insoluble proteins, as well as of water-insoluble'proteins bound on a carrier and of a process for the preparation thereof.

The present invention substantially overcomes the deficiencies of prior carriers and/or techniques for binding proteins to carrier materials by providing novel copolymers eminently utilizable as protein carriers, methods for making same, a technique for binding proteins to such. novel carriers, and the resulting protein/carrier combination as such.

The copolymers according to the present invention essentially consist of (a) acrylamide, together with (b) ethylene-maleic acid or its anhydride and/0r (c) maleic acid, and also (d) N,N-methylene-bis-acrylamide or ethylene diacrylate in a weight ratio azbzczd of 3 0.5 l.5 0.05 4 20.075 0.9, the weight ratio a (b +c) being not more 4.

The Y residue of the dicarboxylic acid in the copolymer, i.e., that derived from component (b) and/or (c), is preferably present in the form of the anhydride. The weight ratio of acrylamide to component (d), i.e., N,N-methylene-bis-acrylamide and/or ethylenediacrylate, is preferably 3 to not more than 0.45.

In the following, the new copolymers according to the present invention, the production thereof and their bonding of the ethylene-maleic acid in the gel.

For use for fixing of proteins, especially good copolymers have been obtained with the use of acrylamide and maleic acid or ethylene-maleic acid or the corresponding anhydrides in a ratio by weight of 3 0.5 to 1.0. In the case of increasing the concentration of the dicarboxylic acid to 1.5, the copolymer becomes more brittle and less swellable but still exhibits good properties as a carrier for proteins. In the case of a ratio of acrylamide to cross-linking agent, i.e., N,N- methylene-bis-acrylamide or ethylene diacrylate, of 3 0.075 to 0.450, there are obtained gel-like polymers with especially good properties. A ratio of 3 0.075 represents the lower limit in which the maleic acid or ethylene-maleic acid can still be satisfactorily incorporated into the copolymer.

For the fixing of proteins in aqueous media, there have also proved to be very useful copolymers with cross-linking agents in amounts corresponding to a weight ratio of acrylamide to cross-linking agent of more than 3 0.45, for example of 3 0.6 0.9. However, when using protein bound to such carriers at salt concentrations of more than 0.05M, the relatively closely cross-linked gels hereby obtained have proved to be disadvantageous for the protein structure.

The production of the copolymers according to the present invention can be carried out, for example, in aqueous solution and preferably in an inert atmosphere. Examples of free radical catalysts or free radical accelerators which can be used include propionic acid nitrile and ammonium peroxydisulphate. The polymerization can be carried out at ambient temperature or also at higher temperatures of up to C., as well as at reduced temperatures. The period of time of the polymerization depends upon the selected polymerization temperature, the selected accelerators or catalysts and on the composition by weight of the reaction mixture. Usually, it is between about 5 minutes and several hours.

After solidification, for the achievement of a particle size which is especially suitable for handling, the polymerized material is forced through a sieve with the desired mesh size, then washed with water and lyophilized.

For the fixing of the protein, it is necessary that the dicarboxylic acid residues in the polymer be present in the form of the anhydride. Therefore, before the bonding of the protein, all acid groups present must again be converted into cyclic anhydride groups. This can be effected, for example, by heating the lyophilized copolymer in a vacuum to a temperature of about 80 C. or at atmospheric pressure to a temperature of about 220C, preferably of 180 200C.

The copolymers according to the present invention are, depending upon the concentration of cross-linking agent, water white to milky cloudy substances with a rubber-like to brittle consistency.

The copolymers can easily be granulated, for example, by forcing them through a metal sieve. Copolymers which are especially suitable for the purpose of protein bonding are those with particle sizes of about 0.35 0.8 mm. Upon the addition of water, the granulate swells. The strength of the swelling is dependent upon the content of cross-linking agent. The more closely the copolymers according to the present invention are cross-linked, the less swellable they are.

The de-swelling can be carried out with the use of, for example, alcohol or, more simply, by lyophilization.

In some respects, the particles of the copolymers according to the present invention are similar to those of the known polyacrylamide molecular sieves. In the case of treatment with buffer solutions, a shrinkage takes place which is dependent upon the number of carboxylic acid groups present, similarly to ion exchangers based on dextran or polyacrylamide.

When the carboxylic acid groups are converted into cyclic anhydride groups by heating to about 200C., a slight yellow coloration frequently occurs. If heating is continued for a comparatively long period of time, then the color deepens from yellow to brown and, at the same time, the binding capacity decreases markedly. Therefore, heating for a comparatively long period of time at the necessary temperature under atmospheric pressure is to be avoided if possible.

The porosity and hardness of the copolymer according to the present invention in the swollen gel state is, in the first place, determined by the content of cross-linking agent. The material exhibits molecular sieve properties, similar to those of polyacrylamide gels, the exclusion limits of which can be determined, as desired, by the content of cross-linking agent. In the swollen state, the copolymer according to the present invention can easily be filtered or centrifuged and has ideal properties for column packing. When swollen in water or in a buffer solution and then de-swollen by lyophilization, the gel-like copolymer is of unlimited life.

The copolymer according to the present invention permits the binding of protein under mild conditions because it is hydrophilic and extremely swellable and the protein bonding only takes place by solvolysis of the cyclic anhydride groups. The protein structure is thus maintained after the bonding of the protein has taken place and activity yields of up to 100 percent and even more, due to a potentiation of the activity, can be obtained. Unbound protein can be recovered without loss of activity. ln the case of the binding, the protein does not perform a cross-linking function in the matrix but rather the carrier-bound enzymes, which can be called enzyme gels," are completely uniform and do not exhibit a differing strength dependent upon the concentration of the protein.

Due to its precise degree of cross-linking, the molecular sieve properties of the copolymer can be so adjusted that only proteins in the desired range can penetrate into it. Thus, it is possible so to cross-link the copolymer for certain proteins that the protein binding can only take place on the surface of the gel particles or, if desired, can also take place in the totality of the gel. The polymerization parameters can be varied according to the effective molecule radii, steric effects and charge distributions. 1

The copolymers according to the present invention possess adsorption properties such that, in water or weak buffer solutions (up to about 0.01M), the protein is completely fixed on the carrier. Up to percent of the protein is hereby covalently bound and the remainder heteropolarly bound. in the case of the use of the copolymers according to the present invention without previous cyclization of the acid groups, the protein bonding only takes place adsorptively. It has been found that, under these conditions, the protein can again be eluted from the carrier with salt solutions, without loss of activity.

The protein-binding capacity of the copolymer according to the present invention is dependent upon the number of anhydride groups present and upon the pore size. In the case of a more closely cross-linked product, the binding capacity is lower than in the case of a less closely cross-linked product with the same number of anhydride groups present since the protein molecule, in the case of closer cross-linking, cannot reach all of the available groups capable of bonding.

lf, in the copolymer according to the present invention, too many charges are present which cannot be occupied by protein by covalent bonding, then generally there takes place a displacement of the pH optima in the case of the bound enzymes. Correspondingly, it has been observed, in the case of known carrier-bound enzymes, that, in the case of negatively charged carried materials, a displacement of two pH units occurred; for example, in the case of trypsin bound to ethylene-maleic acid anhydride, from 7.8 to 10.0 and, in the case of positively charged carriers, a displacement into the acid region took place. ln the case of the copolymers according to the present invention, it is possible to use the groups capable of bonding to a maximum extent so that carrier-bound proteins can be obtained which show no displacement of the pH value optimum.

An especially interesting property of the water-insoluble proteins bound on carriers according to the present invention is that, under certain circumstances, lower Michaelis constants are obtained than in the case of the unbound enzyme. Thus, for example, we have found that the activity of trypsin bound according to the present invention towards N-benzoyl-l-arginine-pnitranilide is increased in comparison with the unbound enzyme.

The carrier-bound enzymes according to the present invention exhibit a plethora of highly desirable properties and can be used for a large number of different fields of application. Thus, they make it possible to carry out substrate reactions with a greater degree of precision than heretofore since the enzyme can again be removed from an incubation batch at any desired point of time. If the reaction is still not quantitative, then the same enzyme can be used again. In the case of proteases, the protein to be split can be isolated at any desired stage of hydrolysis. The fission reaction can thus be followed directly. The enzymatically-active protein must no longer, as was previously the case, be precipitated after the substrate reaction-in this case the substrate reaction product was frequently changed-but can be removed by simple filtering or decanting off or the like.

Substrate reactions can also be carried out in columns with carrier-bound enzymatically active proteins of this type. When the carrier enzyme according to the present invention is packed into a cage-like larly large molecule, then, by reduction of the degree of cross-linking to a large extent, a penetration of the protein intO the matrix and binding thereln can be accomplished. In the case of protein molecules which are vessel, it can be placed in an incubation reaction batch 5 so large that, e en in the case of a cross-linking to the in the manner similar to a tea egg and removed smallest possible extent, their penetration into the therefrom at any time. matrix is substantially impaired, it is also possible to With the use of proteolytically active, carrier-bound carry out a fixing of the protein only on the surface of enzymes, inhibitors can be isolated in a simple manner. the particles, the copolymer in this case being The stability of the proteases is thereby substantially inpreferably of such a fine particle size that a very large creased by the binding to the carrier material since the surface area is obtained so that the fixing of the protein, proteases no longer are subjected to an autolytic reacwhich then only takes place on the surface, results in a tion. completely sufficient degree of fixing.

With carrier-bound protease inhibitors according to the present invention, proteases can be isolated simply, T e at o of homopolar t0 adsorptive or heteropolar quickly and quite specifically. With the use thereof, it is enzymes bound on the carrier can be influenced by the also possible to remove proteases from protease-connature of the pr ion O e bonding between the taining solutions of proteins and thus to stabilize them. protein and thecarrier. If, for example, the protein is By means of antigens and antibodies fixed on carriers present in a relatively high buffer concentration and according to the present invention, there can be carried slowly added to the non-swollen gel, then the yield of out a simple isolation of antigens and anti-bodies. With homopolar-bound enzyme is very high, whereas there is carrier-bound enzyme anti-bodies of this type, an enpractically no adsorptive-bound enzyme. lf, on the zyme isolation can be carried out especially simply and other hand, there is used a lower buffer concentration effectively. and the carrier and enzyme are mixed together quickly,

The bound proteins according to the present inventhen the yield of bound protein is admittedly higher but tion can be used repeatedly and quite generally and a considerable proportion of the protein which has got some of them show no loss of activity even after having on to the carrier can be eluted again with high buffer been used foralong period of tlme. concentrations which, therefore, cannot have been Because of the molecular sieve properties of the homopolar bound. If, for example, as mentioned above, copolymers according to the present invention, the carthere is used a high buffer concentration and slow addirier-bound proteins can also be used for the separation tion of the carrier, then about 80 percent of the enzyme ofsubstrates. present goes on to the carrier and can no longer be The copolymers according to the present invention eluted therefrom. The enzyme is thus substantially are, with regard to their suitability for the fixing of completely homopolarly bound and its activity is solely biologically active proteinS, much superior to previdetermined by the accessibility for the substrate. In the ously known carrier materials used for the same pursecond case, however, in the case of lower buffer conpose, as is shown by the results summarized in the folcentrations and rapid addition, admittedly 100 percent lowing Table l: of the protein present is absorbed on the carrier but up 7 i W 'Tlt'iiiii' Activity 4 Yield, Yield of of low percent hound mol. wt. activity Uniformity of Carrier: protein substrate of starting Carrier material the material protein ratio (in percent) (in percent) material 15-18. 90 10-13. l0 25'30. Up to Up to 14.

EMAJ Nonuniform. :l.. 40-45.... (30-70 Up to 30. According to the invention Uniform As desired 1:2... Up to 80.... Up to 100 Up to 80.

1 lolyacrylamidc.

2 Carboxymethyl cellulose hydrazidc.

3 Ethylene-maleic acid anhydridc.

4 'Irypsin activity.

5 All low molecular weight amino acid substrates. 6 Chymotrypsin activity.

7 Amino acid esters protected on the nitrogen.

5 N bcnzoyl-l-argininc-p nitroanilide.

" The specific copolymer was acrylamide, N,N-mcthylcn-blsncrylamidc and unhytlridc 01 malcic acid.

The superiority of the carrier-bound proteins according to the present invention lies not only in their superior yields and activity but also in the substantially more dependable and definable ratio of the carrier to the protein.

to 35 percent of this can again be eluted with high salt or buffer concentrations and this amount is, therefore, not homopolarly bound.

The following examples are given for the purpose, of illustrating the present invention:

PRODUCTION OF THE COPOLYMERS OF THIS lNVENTlON EXAMPLE 1 3 g. acrylamide and 0.3 g. N,N'-methylene-bisacrylamide were suspended, together with l g. ethylene-maleic acid anhydride, in 23 ml. 0.05M

phosphate buffer of pH 7.6,;and, with vigorous stirring in an atmosphere of nitrogen and at ambient temperature, mixed with 1 ml. of percent propionic acid nitrile and 1 ml. 5 percent ammonium perox ydisulphate. After minutes, the mass solidified EXAMPLE 2 3 g. acrylamide, 0.075 ethylene diacrylate and 1 g. maleic acid were copolymerized in the manner described in Example 1. The period of polymerization was 15 hours. Further working up was carried out in the manner described in Example 1. The yields of lyophilized product was 2.3 g. After cyclization, the weight of the product was 2.05 g.

EXAMPLE 3 3 g. acrylamide, 0.3 g. N,N'-methylene-bis-acrylamide and 1 g. maleic acid were copolymerized in the manner described in Example 1. The period of polymerization was 15 hours. Further working up was also carried out in manner described in Example 1. The yield of lyophilized material was 2.67 g. After cyclization, the weight of the product was 2.45 g.

EXAMPLE 4 3 g. acrylamide, 0.6 g. N,N'-methy1ene-bis-acrylamide and 1 g. maleic acid were copolymerized in the manner described in Example 1. The period of polymerization was 5 hours. Further working up was also carried out in the manner described in Example 1. The yield of lyophilized material was 2.8 g. After cyclization, there was obtained 2.6 g. of end product.

EXAMPLE 5 3 g. acrylamide, 0.3 g. N,N-methylene-bis-acrylamide and 0.1 g. maleic acid were copolymerized for 5 hours in the manner described in Example 1. Further working up was also carried out in the manner described in Example 1. The wet weight of the swollen product was 53.5 g. After lyophilization the dry weight of the product was 4.8 g. and cyclization gave a yield of end product of 3.1 g.

PREPARATION OF CARRIER-BOUND PROTElNS EXAMPLE 6 There was used a cyclized copolymer of 3 g. acrylamide 0.15 g. N,N'-methylene-bis-acrylamide and 1 g. maleic acid as carrier. 100 mg. trypsin were stirred with l g. carrier in 15 ml. ice-cold water (pH 5.2) and allowed to react for 15 hours at 4C. in a cool room. The product was subsequently filled into a column.

The heteropolar bound protein was eluted with 65 ml. wash water. Thereafter, with the use of 0.2M phosphate buffer (pH 7.8), no further protein could be eluted.

Heteropolar-bound protein 31% 'atpl-l 7.0, 8.5, 10 20% Residual activity, referred to non-bound trypsin:

EXAMPLE 7 The product of Example 5 was used as carrier. The carrier was reacted with mg. trypsin in the manner described in Example 6.

Non-bound protein 30% Activity of the bound protein before treatment with 0.2M buffer 100% after treatment with 0.2M buffer 50% EXAMPLE 8 There wasused the same carrier as was employed in Example 6. 100 mg. carrier and 200 mg. trypsin were allowed to react in the manner described in Example 6.

Non-bound protein 50% Activity of the bound protein before treatment with 0.2M buffer 100% after treatment with 0.2M buffer 48% EXAMPLE 9 As carrier, there was used the product of Example 3. 100 mg. calf serum albumin were allowed to react with l g. of the carrier in the manner described in Example 6.

Non-bound protein 32% Protein cannot be eluted from the carrier either with 0.2M phosphate buffer (pH 7.8) or with 0.2M glycine buffer (pH 1.8 The yield of homopolar-bound protein was 68 percent, referred to the amount of protein used, and 100 percent referred to the bound protein.

In the case of the use of human serum albumin instead of calf serum albumin, the same result was obtained.

EXAMPLE 10 The product of Example 3 was used as carrier. 1 g. of carrier was added portionwise, within a period of 60 minutes, to 200 mg. trypsin dissolved in 18 ml. 0.1 M. phosphate buffer (pH 7.8) and allowed to react for 18 hours at 4C.

Non-bound protein 50% Activity of the bound protein, referred to the activity initially used 40%.

No protein and no activity can be eluted from the carrier with 0.2M, 0.33M or 0.5M phosphate buffers.

What is claimed is:

l. A polymer consisting of (a) acrylamide; together with at least one member selected from the group consisting of (b) and (c) wherein (b) is a copolymer of ethylene-maleic acid or its anhydride and (c) is maleic acid or its anhydride', and, in addition, (d) N,N- methylene-bis-acrylamide or ethylene diacrylate; in a weight ratio a:b:c:d of 3 0.5 1.5 0.05 4 0.075 0.9, the weight ratio a (b c) being not more than 4.

2. A polymer as claimed in claim 1 consisting of (1) acrylamide, (2) a copolymer of ethylene-maleic acid or its anhydride, and (3) N,N'-methylene-bis-acrylamide or ethylene diacrylate, in a weight ratio l) 2 (2) 1 (3) of 3 :0.5 L 20.075 -0.9.

3. A copolymer as claimed in claim 1 consisting of (1) acrylamide, (2) maleic acid or its anhydride and (3) N,N'-methylene-bis-acrylamide or ethylene diacrylate in a weight ratio (1) 2 (2) (3) of 3 0.05 4: 0.075 0.9.

4. A polymer as claimed in claim 1 wherein the weight ratio a d is 3 to not more than 0.45.

5. A polymer. as claimed in claim 1, wherein the weight ratio a :d is 3 2 0.6 to 3 :0.9.

6. A polymer as claimed in claim 1 wherein the weight ratio a (b+c) is 3 0.5 to 3 1.0.

7. A copolymer as claimed in claim 1, wherein the dicarboxylic acid residues present are in the form of the anhydride.

8. A polymer as claimed in claim 1 consisting of (a) acrylamide, (b) a copolymer of ethylene-maleic acid or its anhydride, (c) maleic acid or its anhydride, and (d) N,N-methylene-bis-acrylamide or ethylene diacrylate; in a weight ratio azbzczd of 3 0.5 1.5 :0.05 4 0.075 0.9, the weight ratio a (b c) being not more than 4.

U Nrrlcn STA'iES PATENfl (mlrzm. CERTIFICATE QF CQRRECTION Dated Jan. 16,1973

. Inventor) Dieter Jaworek et a1 It is certified that error appears in the aboveddentified patent and that said Letters Patent are hcreby' corrected as shown below:

Front Page In the Title Q For I v "Copolymers of Aeryllamide Gas' Read fNe'w Copolymers and Water Insoluble-Proteins Bound Thereon Signed and sealed this 3rd day of December 1974.

(SEAL) Attest:

'McCQY M. mason JR. 4 1

Attes tjing Officer c. MARSHALL DANN Commissioner of Patents USCOMM-DC ODIN-P6 I 0.3. minim" numue onlct II" 0-in-5:

FORM PO-1050 (o-s9) 

2. A polymer as claimed in claim 1 consisting of (1) acrylamide, (2) a copolymer of ethylene-maleic acid or its anhydride, and (3) N,N''-methylene-bis-acrylamide or ethylene diacrylate, in a weight ratio (1) : (2) : (3) of 3 : 0.5 - 1.5 : 0.075 - 0.9.
 3. A copolymer as claimed in claim 1 consisting of (1) acrylamide, (2) maleic acid or its anhydride and (3) N,N''-methylene-bis-acrylamide or ethylene diacrylate in a weight ratio (1) : (2) : (3) of 3 : 0.05 - 4 : 0.075 - 0.9.
 4. A polymer as claimed in claim 1 wherein the weight ratio a : d is 3 to not more than 0.45.
 5. A polymer as claimed in claim 1, wherein the weight ratio a : d is 3 : 0.6 to 3 : 0.9.
 6. A polymer as claimed in claim 1 wherein the weight ratio a : (b + c) is 3 : 0.5 to 3 : 1.0.
 7. A copolymer as claimed in claim 1, wherein the dicarboxylic acid residues present are in the form of the anhydride.
 8. A polymer as claimed in claim 1 consisting of (a) acrylamide, (b) a copolymer of ethylene-maleic acid or its anhydride, (c) maleic acid or its anhydride, and (d) N,N''-methylene-bis-acrylamide or ethylene diacrylate; in a weight ratio a:b:c:d of 3 : 0.5 - 1.5 : 0.05 - 4 : 0.075 - 0.9, the weight ratio a : (b + c) being not more than
 4. 